Project Summary Agents that activate muscarinic acetylcholine receptors (mAChRs) have exciting potential as novel treatments for the negative symptoms and cognitive disturbances in patients suffering from schizophrenia. Previous mAChR agonists failed in clinical development due to a lack of selectivity for individual mAChR subtypes and adverse effects associated with activation of peripheral mAChRs. We have now discovered and characterized highly selective positive allosteric modulators (PAMs) for individual mAChR subtypes and found that selective PAMs for the M1 mAChR have robust cognition-enhancing effects and may have actions that predict reduction in negative symptoms. Of specific interest was the finding that M1 PAMs enhance a form of M1-dependent long-term depression, termed mLTD, at the hippocampo-prefrontal cortex (PFC) synapse. Impaired LTD at this synapse is disrupted in rodent models of schizophrenia and is thought to correlate with deficits in hippocampo-PFC communication observed in schizophrenia patients. It was assumed that M1 PAMs act through potentiation of canonical G?q signaling, which leads to the activation of phospholipase C (PLC) and that activation of PLC is a key mechanism by which M1 activation induces multiple CNS effects. However, our lab and others have shown that M1 activation can also lead to activation of phospholipase D (PLD), independent of PLC activation. While most M1 PAMs potentiate coupling of M1 to both PLC and PLD, we reported the discovery of a novel M1 PAM that displays stimulus bias and thereby potentiates M1 coupling to PLC activation, while having no effect on PLD activity. I now present exciting data using novel highly selective PLD inhibitors suggesting that activation of PLD is required for induction of mLTD in the PFC. Whether potentiation of M1 coupling to PLD is required for M1 PAM-induced reversal of synaptic plasticity and behavioral deficits in a rodent model of cortical dysfunction in schizophrenia remains unknown. Completion of this research proposal will elucidate new basic biology and signaling downstream of the M1 mAChR while also providing key insight into the therapeutic potential of allosteric modulation of M1 as a potential treatment for the cognitive and negative symptoms observed in patients with schizophrenia.